The mechanisms regulating transepithelial permeability, specifically paracellular permeability (flux of solutes between rather than through cells), have become an intensively investigated area in epithelial physiology. Paracellular permeability is controlled at the tight junctional (TJ) band which surrounds each cell of the epithelial sheet (Figure 1). Our previous work has shown that protein kinase C (pKC) activation increases TJ permeability within minutes. This effect was most prominent when activating PKC with the phorbol ester tumor promoters in acute exposure of cell sheets lasting up to several hours. Although this acute action on TJ permeability was increasing our understanding of TJ regulation, its implications for epithelial carcinogenesis and transformation were unclear. We have therefore shifted our focus to chronic exposure of cell sheets to phorbol eaters, now patterning our experiments along the mouse skin studies which defined the nature of tumor promoters. Treatment with phorbol eaters for as long as 18 weeks has yielded three significant findings: 1) the TJs of cell sheets steadily recover their barrier capability over the course of 6 weeks of phorbol ester exposure, the opposite of the effect of acute exposure; 2) PKC is translocated and down regulated during acute exposure, then a new isoform appears as barrier capability is recovering and chronic exposure continues; and 3) the barrier abruptly becomes more leaky again at 6 - 8 weeks of exposure, and simultaneously, polyp-like overgrowths (PLOs) appear across the cell sheet. The emergence of the PLOs consistently at the 6-8 weeks of phorbol ester exposure, and the fact that the TJs between cells of the PLOs are the point of greatest permeability across the cell sheet suggest that: a) we have produced and begun to characterize a potentially highly useful in vitro model for epithelial transformation; b) tight junction permeability does play a role in epithelial transformation; and c) these physiological changes may be caused by biochemical changes in the specific isoform expression of PKC. Our research plan to pursue these hypotheses is now formulated as follows: 1) further characterize the PLOs (relative to adjacent normal epithelium) by examination of their TJ permeability, their cell polarity, the phosphorylation state and localization of their TJ proteins, their overall PKC activity and specific isoform content, their degree of transformation and genetic stability; 2) determine the effect on the PLO properties of first treating of cells with a primary "initiator" carcinogen before the chronic phorbol ester exposure; 3) determine the effect of overexpression of specific isoforms of PKC (by stable and transient transfections) on TJ permeability and PLO formation; and 4) test the human relevance of our model and hypotheses using human colon surgical specimens.